, MBA4, has been isolated for its ability to grow on monobromoacetate (MBA) [8]. This bacterium produces MEK inhibitor clinical trial a haloacid dehalogenase that allows the cell to grow on MBA. Since MBA is a more potent mutagen than ethylmethane sulfonate [9] one would not expect an uptake mechanism for this kind of compound. We have, however, identified a haloacids-transporter protein gene downstream of the dehalogenase gene. This haloacid permease, Deh4p, was expressed, together with the dehalogenase, to enhance the uptake of haloacetates [10]. The gene encoding for Deh4p has been cloned and expressed in E. coli which

facilitated the specific uptake of haloacetates [11]. Deh4p is 552 residues long and has a putative molecular weight of 59,414 and an isoelectric point of 9.14. With the blooming of the sequencing data and the development of bioinformatics, ICG-001 in vitro software that predicts the structure of a protein has become more and more readily available [12–21]. Topology prediction programs that use different algorithms are easily accessible from the Internet

and their predictions are becoming www.selleckchem.com/products/R788(Fostamatinib-disodium).html more and more accurate. Comparative analysis of the primary structure of Deh4p with proteins in the Pfam database [22] has designated it as a member of the Major Facilitator Superfamily [23] (MFS, TC 2.A.1). MFS is a major class of membrane transporter with more than a thousand known proteins [24]. It is also described second as the uniporter-symporter-antiporter family. Although there are many members in this family, only four of them have well defined structure or topology. These proteins are EmrD [25], LacY [26] and GlpT [27], all from Escherichia coli and OxlT from Oxalobacter formigenes [28, 29]. They have been shown to possess twelve transmembrane segments (TMS) with a 2-fold symmetry roughly dividing the first and the second 6-TMS. The termini of these proteins were found to reside within the cytoplasm. Though MFS transporters with 14 and 24 TMS are known [30, 31], they are relatively few in number [32]. Hence the presence of twelve TMS was believed to be the standard characteristic of the MFS proteins. Notwithstanding

the abundance and improved accuracy of those computer analysis methods, experimental determination is still necessary. The use of reporter fusion analyses is by far the most convenient method and the use of dual-reporters is no doubt a better choice than the use of a single indicator [33, 34]. Here we report the experimental determination of the topology of Deh4p using a PhoA-LacZ dual-reporters system [33] and the verification using a comparative approach. Results Hydropathy analysis of Deh4p Computational analysis of Deh4p has categorized it as a MFS protein. This classification was based on the following grounds. First, Pfam [22] analysis (accessed on 29 May, 2009) indicated that Deh4p is a member of the clan MFS and has a signature of PF00083 sugar (and other) transporter family.